The overall goal of my project is to engineer proteins that perturb and thereby elucidate the molecular mechanisms by which adhesion G protein-coupled receptors (aGPCRs) function. Members of the superfamily of cell-surface signaling proteins known as G protein-coupled receptors (GPCRs) have been validated as successful drug targets. aGPCRs make up the second largest GPCR family and have recently been implicated in a variety of diseases, including neurological disorders, and many types of cancer. Characterized by their large and diverse extracellular regions (ECRs), aGPCRs play a role in cell adhesion. Due to the importance of aGPCRs in disease, as well as the proven druggability of GPCRs, aGPCRs are an unexplored, yet potentially impactful class of drug targets. To this end, before drug development can begin, it is imperative to systematically define the feasibility of targeting aGPCRs with drugs. One of the best-studied aGPCRs, GPR56, has been linked to the progression of many cancers as well as brain development, including its role in the developmental brain disorder bilateral frontoparietal polymicrogyria. Though two GPR56 ligands have been identified, the molecular mechanisms by which GPR56 functions remain unclear. Key to my project is the synergistic implementation of GPR56 fragment purification, cutting-edge protein engineering, X-ray crystallography, and cell signaling assays. Using these techniques, I will systematically elucidate the molecular mechanisms that govern the biological functions of GPR56 and thereby assess its durggability. My proposal consists of three aims, the first of which is to engineer binding proteins, termed monobodies (Mbs), that specifically bind GPR56 with high affinity. Using purified GPR56 fragments as targets for phage display library selection, I have engineered a Mb that binds the ECR of GPR56 with high affinity and specificity. Using the techniques I have already successfully employed, I will engineer Mbs that bind other sites on GPR56. Second, I will determine the regions of GPR56 that interact with natural and engineered proteins. I have solved the crystal structure of my Mb bound to the ECR of GPR56. This is the first structure of the full ECR of any aGPCR and also the first structure of an aGPCR fragment in complex with a binding partner. I will crystallize soluble fragments of GPR56 in complex with new Mbs in order to precisely map their binding sites. I will also purify tissue transglutaminase 2, a ligand of GPR56, and attempt to solve its structure bound to GPR56. Third, I will characterize the biological roles of distinct regions of GPR56. By establishing GPR56 signaling assays in my lab, I will assess the functional implications of Mb binding and structure-guided GPR56 mutations in parallel. With the ultimate goal of combatting aGPCR-mediated diseases, it is crucial to gain a clear and accurate understanding of the mechanisms that govern GPR56 function such that the feasibility of drugging this important aGPCR can be evaluated.